Method of preparing polynucleotide-carrier complexes for delivery to cells

Abstract

An improved method of forming substantially disperse and homogeneous polynucleotide-carrier complexes is disclosed. The polynucleotide-carrier complexes can be administered in vivo to obtain significant levels and duration of gene expression.

What is claimed is: 
     
       1. A method of forming a composition of polynucleotide-carrier complexes comprising: (a) preparing a carrier solution comprising cationic carrier molecules, a charge shielding agent in an amount sufficient to inhibit aggregation of the carrier molecules, and a charge neutralizing agent in an amount sufficient to neutralize a portion of the positive charge of the carrier; and   (b) combining the carrier solution with a polynucleotide, thereby forming a solution of polynucleotide-cerrier complexes.   
     
     
       2. The method of claim 1 wherein the charge neutralizing agent is a base. 
     
     
       3. The method of claim 2 wherein the base is selected from the group consisting of sodium hydroxide (NaOH), potassium hydroxide (KOH), ammonium hydroxide (NH 4  OH), alkylamines, alkoxides and triethanolamines. 
     
     
       4. The method of claim 3 wherein the base is sodium hydroxide (NaOH). 
     
     
       5. The method of claim 4 wherein the concentration of base in the carrier solution is about 10-1000 mM. 
     
     
       6. The method of claim 5 wherein the concentration of carrier molecules in the carrier solution is about 0.5 mg/mL-20.0 mg/mL. 
     
     
       7. The method of claim 1 wherein the solution of polynucleotide-carrier complexes has a final concentration of charge shielding agent and charge neutralizing agent which does not damage or substantially inhibit the function of the polynucleotide. 
     
     
       8. The method of claim 7 wherein the charge shielding agent is a salt and the charge neutralizing agent is a base. 
     
     
       9. The method of claim 8 wherein the final salt concentration is about 0.15-0.5 M and the final base concentration is about 1.0-4.0 mM. 
     
     
       10. The method of claim 9 wherein the final salt concentration is about 0.3 M and the final base concentration is about 2.0 mM. 
     
     
       11. A method of forming a composition of polynucleotide-carrier complexes comprising: (a) preparing a carrier solution comprising cationic carrier molecules, a salt in an amount sufficient to inhibit aggregation of the carrier molecules, and a base in an amount sufficient to neutralize a portion of the positive charge of the carrier; and   (b) combining the carrier solution with a polynucleotide at a final salt concentration which does not damage or substantially inhibit the function of the polynucleotide, thereby foaming a solution of polynucleotide-carrier complexes.   
     
     
       12. The method of claim 11 wherein the portion is about 5-20%. 
     
     
       13. The method of claim 11 wherein the carrier solution has a concentration of carrier molecules of about 3.0-7.0 mg/mL, a concentration of base of about 10-100 mM, and a concentration of salt of about 1.0-5.0 M, and wherein the solution of polynucleotide-carrier complexes has a final concentration of base of about 1.0-4.0 mM and a final concentration of salt of about 0.15-0.5 M. 
     
     
       14. The method of claim 13 wherein the solution of polynucleotide-carrier complexes has a final concentration of base and salt of about 2.0 mM and about 0.3 M, respectively. 
     
     
       15. The method of claim 13 wherein the base is sodium hydroxide (NaOH). 
     
     
       16. A method of forming a composition of polynucleotide-carrier complexes comprising: (a) preparing a carrier solution comprising cationic carrier molecules at a concentration of about 3.0-7.0 mg/mL, a salt at a concentration of about 1.0-5.0 M, and a base at a concentration of about 10-100 mM; and   (b) combining the carrier solution with a polynucleotide at a final salt concentration of about 0. 15-5.0 M and a final base concentration of about 1.0-4.0 mM, thereby forming a solution of polynucleotide-carrier complexes.   
     
     
       17. The method of claim 16 wherein the salt is sodium chloride (NaCl) and the base is sodium hydroxide (NaOH). 
     
     
       18. The method of claim 17 wherein the carrier solution has a carrier molecule concentration of about 5.6 mg/mL, a salt concentration of about 4.7 M, and a base concentration of about 59 mM, and wherein the solution of polynucleotide-carrier complexes has a final salt concentration of about 0.3 M, and a final base concentration of about 2.0 mM. 
     
     
       19. The method of claim 16 further comprising the step of extruding the complex and recovering the extruded product. 
     
     
       20. A method of forming a composition of carrier molecules comprising preparing a solution comprising cationic carrier molecules, a salt at a concentration of about 1.0 M or more, and a base in an amount sufficient to neutralize a portion of the positive charge of the cationic carrier molecules. 
     
     
       21. A solution of carrier molecules for forming polynucleotide-carriage complexes, the solution comprising cationic carrier molecules, a charge shielding agent at a concentration of about 1.0 M or more, and a charge neutralizing agent in an amount sufficient to neutralize a portion of the positive charge of the carrier molecules. 
     
     
       22. The solution of claim 21 wherein the charge neutralizing agent is a base. 
     
     
       23. The solution of claim 22 wherein the cationic carrier molecules are at a concentration of about 0.5-20 mg/mL, wherein the charge shielding agent is a salt at a concentration of about 1.0-10 M, and wherein the charge neutralizing agent is a base at a concentration of about 10-1000 mM. 
     
     
       24. The solution of claim 23 wherein the cationic carrier molecules are at a concentration of about 3.0-7.0 mg/mL, wherein the salt is at a concentration of about 1.0-5.0 M, and wherein the base is at a concentration of about 10-100 mM. 
     
     
       25. The solution of claim 24 wherein the cationic carrier molecules are at a concentration of about 5.0-6.0 mg/mL, wherein the salt is at a concentration of about 4.0-5.0 M, and wherein the base is at a concentration of about 50-70 mM. 
     
     
       26. A solution comprising substantially unaggregated polynucleotide-carrier complexes, a charge shielding agent at a concentration of about 0.5 M or less, and a charge neutralizing agent at a concentration of about 10 mM or less. 
     
     
       27. The solution of claim 26 wherein the charge shielding agent is a salt at a concentration of about 0.15-0.5 M and the charge neutralizing agent is a base at a concentration of about 1.0-4.0 mM. 
     
     
       28. The solution of claim 27 wherein the polynucleotide complexes have been extruded.